One of the goals of the Integrative Neuroscience Initiative on Alcoholism (INIA)-Neuroimmune consortium is to understand brain immune signaling systems and their role in the causes and treatments of alcohol dependence. As part of INIA, we have identified the receptor tyrosine kinase, anaplastic lymphoma kinase (ALK) and its putative secreted ligand midkine (MDK), as novel signaling proteins that may be involved in alcohol use disorders. ALK and MDK are known to signal through immune response pathways in the body, but the roles of ALK and MDK in immune signaling specifically in the brain in response to ethanol have not been examined. The objective of the first Specific Aim of this project is to identify ethanol-responsive MDK- and ALK-dependent neuroimmune signaling pathways in the brain using MDK and ALK inhibitors. This will be done by treating mice either with a single dose of ethanol or allowing mice to drink alcohol. Phosphorylation of proteins involved in neuroimmune signaling pathways will be measured in the first experiment. In the second experiment, expression of genes encoding cytokines and chemokines will be tested. In the third experiment, activation of microglia, the resident immune cells in the brain, and astrocytes, which are activated by inflammatory processes, will be examined. Small-molecule inhibitors targeting ALK are effective in reducing binge-like drinking in mice, suggesting that ALK is a potential therapeutic target for the treatment of alcohol use disorders. The mechanism(s) through which ALK acts to regulate excessive drinking are not well understood. Evidence from our INIA work suggests that ALK might act in the central nucleus of the amygdala (CeA), a key brain region involved in ethanol dependence, to regulate excessive ethanol consumption and GABA neurotransmission. The goal of the second Specific Aim of this project is to test for effects of MDK and ALK manipulation in the CeA on these two parameters. Three experiments will be performed. The first involves viral delivery of short hairpin RNAs targeting ALK or MDK directly into the CeA. In the second and third experiments, we will infuse ALK and MDK inhibitors, or recombinant MDK protein, into the CeA. Mice will be tested for binge-like ethanol consumption and for GABA-mediated inhibitory currents by electrophysiology after infusion. Another goal of INIA-Neuroimmune is to use new technologies to study neural circuits involved in the development of excessive alcohol drinking. To this end, the objective of the third Specific Aim is to develop and produce cutting-edge viral vectors for INIA investigators for selective knockdown, knockout, or overexpression of genes. These vectors will be provided to INIA investigators for delivery to specific brain regions involved in alcohol dependence. This will allow for the identification of the neural pathways in which INIA candidate genes function to control alcohol drinking. This proposal involves numerous interactions with INIA-Neuroimmune investigators and will assist in the achievement of the goals of the consortium.